Inactivated rabies vaccine for veterinary use

ABSTRACT

The preparation of a vaccine for immunization of canine and feline animals from inactivated HCP-SAD strain of rabies virus is disclosed.

This invention relates to veterinary rabies vaccines. More particularly, the invention relates to the propagation of rabies virus in swine testicle cell cultures, to mono- and polyvalent vaccines containing the inactivated rabies virus so propagated and to the use of such vaccines to vaccinate canine and feline animals. The inactivated rabies vaccine of this invention is particularly useful for vaccinating dogs and cats and produces advantageously high antibody responses in cats.

For many years, research has been directed toward the preparation of safe and effective veterinary rabies vaccines [Crick et al., Vet. Rec. 99(9):162 (1976); Plotkin et al., Ann. Rev. Med. 29:583 (1978)]. A number of rabies vaccines are currently marketed for use in dogs, cats and other animals. These vaccines are classified as nervous tissue vaccines, avian embryo vaccines and tissue culture vaccines, depending on the medium in which the virus was propagated. The inactivated rabies vaccines currently marketed are of murine (nervous tissue) and hamster cell line origin (tissue culture) [Compendium of Animal Rabies Vaccines, 1980, J. Amer. Vet. Med. Assoc. 176(5):399 (1980)] and are known to be of limited safety, particularly in use with cats. In fact, high cell passage SAD rabies strain vaccines are no longer approved by the United States Department of Agriculture for vaccination of cats.

Many known veterinary rabies vaccines comprise virus which has been propagated in tissue culture. For example, the Flury HEP strain was grown in canine kidney cell [Brown et al., Amer. J. Vet. Res. 28(124):751 (1967)], the ERA strain was propagated and attenuated in porcine kidney cells (U.S. Pat. No. 3,423,505), the PRI strain was produced by repeated passage of the ERA strain in porcine kidney cells (U.S. Pat. No. 4,040,904) and the ERA strain has been attenuated in bovine kidney cells (U.S. Pat. No. 3,585,266; German Pat. No. 2,162,013). Other cells used for the production of attenuated or inactivated rabies vaccines include hamster fibroblasts [C. R. Hebd. Seances Acad. Sci. Ser. D. Sci. Natur. 265(25):2143 (1967)], baby hamster kidney cells [Crick et al., Res. Vet. Sci. 12(2):156 (1971); U.S. Pat. No. 3,769,415], chick embryo fibroblasts (U.S. Pat. No. 4,115,195; Belgian Pat. No. 863,368), fetal calf kidney cells (French Pat. Nos. 2,261,779 and 2,290,220), fetal canine lung diploid cells (Belgian Pat. No. 859,178), human diploid cells (U.S. Pat. No. 3,397,267), a diploid porcine embryonic cell strain (U.S. Pat. No. 4,070,453), human and murine neuroblastoma cells [Clark, Science 199(4333):1072 (1978) and Infect. Immun. 27(3):1012 (1980)], African green monkey kidney cells [Nawathe et al., Bull. Anim. Health Prod. Afr. 26(1):1 (1978)] and quail embryo primary cells [Bektemirova et al., Arch. Virol. 61(1-2):61 (1979)].

Until the present work, rabies virus has not been adapted for growth in swine testicle cell cultures. The present invention consists of the growth of rabies virus in swine testicle cell cultures, particularly in a diploid swine testicle cell line designated the NL-ST-1 cell line, and the preparation of safe and highly effective mono- and polyvalent vaccines from the inactivated virus for immunization of canine and feline animals against rabies. Swine testicle cell cultures used to propagate the rabies virus are described by McClurkin et al., Can. J. Comp. Med. Vet. Sci. 30:190 (1966). Use of the NL-ST-1 cell line for virus production was approved by the United States Department of Agriculture, Animal and Plant Health Inspection Service in November, 1976; a pseudorabies vaccine containing virus propagation on this cell line was licensed and marketed in the United States in 1977.

The monovalent vaccine of this invention is administered parenterally, preferably by intramuscular injection, in one or more doses. Preferably, a single 1.0 ml to 1.2 ml dose of vaccine containing 0.93 ml of inactivated virus-containing fluids having from about 10⁴.0 to about 10⁹.0 TCID₅₀ /ml, preferably from about 10⁶.0 to about 10⁸.0 TCID₅₀ /ml, combined with a suitable carrier, adjuvant and/or stabilizer is administered. Animals younger than three months of age when initially vaccinated should be revaccinated after reaching the age of three months. Annual revaccination is recommended.

The rabies virus used to prepare the inactivated virus vaccine of this invention is the high cell culture passage of the Street Alabama Dufferin (HCP-SAD) rabies virus strain. This virus was initially isolated from a rabid dog at CDC-Dufferin Laboratories, Montgomery, Alabama in 1935. The isolate was passaged 54 times in mice, followed by 25 passages in hamster kidney cell culture, 10 passages in embryonated chicken eggs and 40 serial passages in porcine kidney cell culture. The virus is then adapted for growth in the swine testicle cell cultures by passaging at least once. The virus may be passaged up to about 25 times in swine testicle cell cultures, with from about 6 to about 12 passages being preferable. Before passaging in swine testicle cells, the virus may be passaged in other mamalian cell cultures such as bovine kidney cell cultures.

After growth in swine testicle cells at from about 34° C. to about 38° C., preferably about 36° C., the virus is inactivated with an inactivating agent which does not destroy the virus particles or antigenicity according to standard methods known to the art. Examples of such inactivating agents are beta-propiolactone or ethyleneimine derivatives, preferably beta-propiolactone.

To prepare the vaccine of this invention, the inactivated rabies virus is combined with an adjuvant, a suitable carrier and/or a stabilizer according to standard, known to the art methods. Any known adjuvant which enhances the antigenicity of the vaccine, for example aluminum hydroxide gel, may be used.

It has been found that a single 1.0 ml intramuscular vaccination with the inactivated rabies vaccine of this invention having a titer of about 10⁶.0 to about 10⁸.0 TCID₅₀ /ml elicited significant serological responses in 100% of vaccinated dogs. At one year following vaccination, 96% of these dogs remained protected against challenge with virulent virus which killed 100% of the unvaccinated controls. In cats, vaccination with a single 1.0 ml intramuscular vaccination of the inactivated rabies vaccine of this invention having a titer of about 10⁶.0 to about 10⁸.0 TCID₅₀ /ml produced a surprising and highly significant serological response in 100% of the vaccinated animals. At one year following vaccination, 100% of these cats remained protected against challenge with virulent virus which killed 90% of the unvaccinated controls. Thus, the inactivated rabies vaccine of the present invention is a safe and effective veterinary vaccine, being particularly safe, effective and superior to prior art vaccines for protecting felines against rabies.

The inactivated rabies vaccine of this invention has been licensed for use in dogs and cats by the United States Department of Agriculture, Animal and Plant Health Inspection Services on June 16, 1980. It is believed to be the only vaccine containing the SAD strain of rabies virus approved for use in cats.

DETAILED DESCRIPTION OF THE INVENTION Preparation of the Inactivated Rabies Vaccine

The high cell passage of Street Alabama Dufferin (HCP-SAD) strain of rabies virus was used to prepare the inactivated rabies vaccine of this invention. This virus was obtained from a commercial rabies vaccine manufactured by Jensen-Salsbury Laboratories, Kansas City, Missouri at the 129th passage level and was further transferred for 15 additional serial passages in a bovine kidney cell culture and 6 serial passages in the swine testicle cell line, NL-ST-1. The 150th passage was designated as the master seed virus. The virus was identified by specific immunofluorescence of infected cell cultures stained with fluorescein conjugated rabies specific antiserum. Viral antigen was demonstrated in the cytoplasm of infected cells. The virus was also identified by neutralization with specific rabies antiserum. Virulence was demonstrated by intracerebral inoculation of the master seed virus into young adult mice.

For propagation of the master seed virus, frozen ampoules of NL-ST-1 cells were thawed and reconstituted to sufficient volume for seeding in production flasks. The production flasks were allowed to incubate at 36° C. until a confluent cell monolayer formed (3 to 5 days). Passage of these cells was accomplished by use of a combination of versene-trypsin and the cells were expanded in volume to plant sufficient numbers in production flasks for inoculation. While the cells were suspended in the growth medium, master seed virus fluids were added to the suspension at 0.1% of total medium volume. Production flasks were planted with the virus-inoculated growth medium and the cells allowed to multiply until 90-100% confluency of cell monolayer was achieved (3 to 5 days). These virus-infected cells were removed from the production flasks with versene-trypsin and suspended in maintenance medium. Production flasks were planted with maintenance medium containing the virus-infected cell suspension and maintained at 36° C. until harvest. Replication of the rabies virus is complete at the time of appearance of cytopathogenic effects, characterized by rounded cells which subsequently become detached from the cell monolayers. Contamination was detected by gross and microscopic examination. Titer of the master seed virus inoculum was at least 10⁶.0 TCID₅₀ /ml.

Harvest of virus-laden fluids following production flask planting, which is dependent upon the cytopathogenic changes occurring in the infected monolayer, occured after 3 to 6 days. The virus-laden fluid was transferred and pooled into sterile storage containers under sterile environmental conditions. Samples of harvested fluids were tested for sterility and virus titer. Only fluids found to be pure were used for vaccine preparation. Virus titer of the harvested fluids must be from about 10⁴.0 to about 10⁹.0 TCID₅₀ /ml.

To inactivate the harvested virus, beta-propiolactone was added to the pooled fluids at pH 6.8 to 7.4 to a final concentration of 1:6000 (0.017%). The virus containing fluids were maintained at 4° C. with constant stirring for 48 hours. Following inactivation, merthiolate (thimersol, N.F.) was added as a preservative to give a final concentration of 1:10,000. After the addition of merthiolate, the product was stored at 4° C. until addition of adjuvant.

The inactivated virus fluids were pooled, the pH was adjusted to 6.0 with 4 N HCl and aluminum hydroxide gel (2% Al₂ O₃) was added as adjuvant until a final concentration of 5% by volume. The inactivated virus-aluminum hydroxide gel mixture was stirred for 2 hours at 4° C. The vaccine thus prepared was tested for safety and potency and stored at 4° C.

An accelerated stability study was conducted to determine stability of the vaccine. After incubation of samples of a vaccine prepared as described above at 37° C. for one week, there was a loss of 0.04 relative potency as compared with a sample stored at 4° C.

The relative potency of the vaccine was determined by the National Institutes of Health (NIH) mouse potency test described in Chapter 33 of "Laboratory Techniques in Rabies", 3rd edition, WHO, Geneva (1973) by comparing test results of five samples of the vaccine to test results of five samples of NIH standard reference vaccine. The geometric mean relative potency value was 0.15.

Use of the Inactivated Rabies Vaccine

The inactivated rabies virus vaccine was used to vaccinate susceptible dogs and cats. Rabies-susceptible dogs were obtained from commercial sources and ranged in age from 4.5 to 7.5 months when vaccinated. Rabies-susceptible cats were obtained from commercial sources and ranged in age from 7 to 13 months when vaccinated. Each animal was vaccinated intramuscularly at one site in the thigh with a single 1 ml dose of vaccine having a titer of 10⁶.4 TCID₅₀ /ml, either undiluted (full dose) or diluted 1:2 (half dose). The half dose vaccine was prepared by mixing equal amounts of the undiluted vaccine and diluent containing 5% aluminum hydroxide gel. All vaccinated animals were bled prior to vaccination and at 30, 60, 90, 180, 270 and 365 days following vaccination. Serum was collected and evaluated for the presence of serum neutralizing rabies antibody in accordance with the Quantitative Assay and Potency Test of Antirabies Serum and Immunoglobulin in Chapter 40 of "Laboratory Techniques in Rabies", 3rd edition, WHO, Geneva (1973). Serum-neutralization (SN) antibody titer was determined by the method of Reed and Muench, J. Amer. J. Hygiene 27:493 (1938).

At 365 days following vaccination, the immunity of the test animals was challenged with virulent NYC strain of street rabies virus. The challenge virus was a dilution of a 20% fox brain suspension.

Vaccination and Challenge of Dogs

Twenty-six dogs determined to be serologically negative to rabies virus were vaccinated with a full dose of the inactivated rabies vaccine. At 30 days following vaccination, the sera of the vaccinates contained a geometric mean antibody titer of 1:109, with titers ranging from 1:32 to 1:431. During a one year serological evaluation, titers gradually declined to a geometric mean antibody titer of 1:10 at 365 days following vaccination. Twenty susceptible control dogs were inoculated with virulent NYC strain of street rabies virus, ten animals receiving virus diluted 1:150 and ten receiving virus diluted 1:300. One year (365 days) following vaccination, the vaccinated dogs were challenged with a 1:150 dilution of the virulent rabies virus (NYC strain of street rabies virus). Twenty-five out of twenty-six (96%) of the vaccinates remained normal throughout the 90 day period following challenge. The results from this test appear in Table 1; results from the control animals appear in Table 3.

Twenty-five dogs determined to be serologically negative to rabies virus were vaccinated with a half dose (1:2) of the inactivated rabies vaccine. At 30 days following vaccination, the sera of the vaccinates contained a geometric mean antibody titer of 1:42, with titers ranging from 1:8 to 1:204. Titers gradually declined to a geometric mean antibody titer of 1:7 at 365 days following vaccination. The immunity of the animals was challenged with virulent NYC strain of street rabies virus 365 days following vaccination. Twenty-three out of twenty-five (92%) of the vaccinated dogs survived challenge. The results from this test appear in Table 2; results from the control animals appear in Table 3.

Vaccination and Challenge of Cats

Twenty-five cats determined to be serologically negative to rabies virus were vaccinated with a full dose of the inactivated rabies vaccine. At 30 days following vaccination, the sera of the vaccinates contained a geometric mean antibody titer of 1:1328, with titers ranging from 1:214 to 1:8192. At 365 days following vaccination, the geometric mean antibody titer was 1:88 with titers ranging from 1:26 to 1:1024. As controls, twenty susceptible cats were inoculated with virulent NYC strain of street rabies virus, ten animals receiving virus diluted 1:30 and ten receiving virus diluted 1:60. The immunity of the vaccinated cats was challenged at 365 days following vaccination with virulent rabies virus (NYC strain of street rabies virus). All (100%) of the vaccinated cats remained normal throughout the 90 day period following challenge. The results from this test appear in Table 4; results from the control animals appear in Table 5.

                  TABLE 1                                                          ______________________________________                                         Protection Afforded Dogs by Vaccination with Inactivated                       Rabies Vaccine, Swine Testicle Cell Line Origin (Full Dose)                          Serum Neutralization Titer* Following                                                                  Status                                           Dog   Vac. (days)             Post                                             No.   0      30     60   90  180  270  365  Challenge                          ______________________________________                                         10    Neg.   81     16   8   4    13   64   Normal                             11    Neg.   38     5    4   3    Neg. Neg. Normal                             16    Neg.   38     41   13  5    2    2    Normal                             20    Neg.   206    19   8   3    5    10   Normal                             22    Neg.   128    10   4   3    4    13   Died (Rabies,                                                                  Day 19)                            23    Neg.   128    54   64  19   19   2    Normal                             27    Neg.   64     76   81  54   64   51   Normal                             29    Neg.   203    41   19  25   32   38   Normal                             31    Neg.   203    41   65  54   25   32   Normal                             45    Neg.   431    12   11  13   5    5    Normal                             62    Neg.   128    5    4   6    2    4    Normal                             79    Neg.   98     5    8   13   25   16   Normal                             82    Neg.   87     214  54  13   11   10   Normal                             83    Neg.   431    13   64  76   64   128  Normal                             94    Neg.   182    19   19  19   19   54   Normal                             97    Neg.   203    25   21  19   13   6    Normal                             98    Neg.   38     16   64  16   6    3    Normal                             99    Neg.   203    54   16  16   10   6    Normal                             100   Neg.   32     5    54  25   13   13   Normal                             101   Neg.   256    19   54  19   16   10   Normal                             107   Neg.   81     3    3   4    4    10   Normal                             112   Neg.   38     10   54  6    4    2    Normal                             144   Neg.   81     128  46  13   10   19   Normal                             145   Neg.   107    20   16  6    16   10   Normal                             154   Neg.   81     41   6   2    Neg. 2    Normal                             155   Neg.   85     32   19  2    3    2    Normal                             Geo.                                                                           Mean  Neg.   109    21   19  10   10   10                                      ______________________________________                                          *Titer expressed as reciprocal of serumneutralization end point.         

                  TABLE 2                                                          ______________________________________                                         Protection Afforded Dogs by Vaccination with Inactivated                       Rabies Vaccine, Swine Testicle Cell Line Origin (Half Dose)                          Serum Neutralization Titer* Following                                                                  Status                                           Dog   Vac. (days)             Post-                                            No.   0      30     60   90  180  270  365  Challenge                          ______________________________________                                         2     Neg.   8      3    5   5    3    2    Normal                             3     Neg.   20     4    2   4    5    3    Normal                             4     Neg.   204    54   25  13   6    16   Normal                             19    Neg.   20     5    5   4    5    3    Normal                             21    Neg.   81     5    10  4    4    3    Normal                             42    Neg.   51     41   10  3    2    Neg. Normal                             43    Neg.   151    46   25  5    4    3    Normal                             64    Neg.   32     5    14  8    13   4    Normal                             67    Neg.   27     Neg. 2   5    3    Neg. Normal                             74    Neg.   32     6    3   Neg. 6    3    Normal                             78    Neg.   16     2    2   2    2    4    Normal                             81    Neg.   32     3    3   13   6    10   Normal                             103   Neg.   13     4    3   4    3    4    Normal                             104   Neg.   13     22   10  Neg. 3    2    Died (Rabies,                                                                  day 14)                            108   Neg.   51     13   6   13   10   32   Normal                             109   Neg.   51     11   8   4    4    5    Normal                             110   Neg.   32     5    5   3    3    4    Normal                             111   Neg.   28     4    2   3    Neg. Neg. Normal                             115   Neg.   38     10   3   8    6    6    Died (Rabies,                                                                  day 14)                            118   Neg.   166    32   41  13   20   21   Normal                             123   Neg.   81     41   25  25   11   54   Normal                             125   Neg.   81     13   3   Neg. 4    Neg. Normal                             126   Neg.   27     13   16  13   10   13   Normal                             143   Neg.   128    19   19  16   20   19   Normal                             147   Neg.   151    16   41  41   102  54   Normal                             Geo.                                                                           Mean  Neg.   42     10   7   7    6    7                                       ______________________________________                                          *Titer expressed as reciprocal of serumneutralization end point.         

                  TABLE 3                                                          ______________________________________                                         Dog Controls - 20% Fox Brain Suspension of NYC Strain of                       Street Rabies Virus (0.5 ml Bilateral Masseter Muscles)                        Challenge      Dog.                                                            Dilution       No.        Observations                                         ______________________________________                                         1:150          70         Rabies - Day 11                                                     71         Rabies - Day 10                                                     87         Rabies - Day 10                                                     106        Rabies - Day 10                                                     116        Rabies - Day 10                                                     130        Rabies - Day 13                                                     132        Rabies - Day 12                                                     135        Rabies - Day 13                                                     136        Rabies - Day 10                                                     137        Rabies - Day 14                                      1:300          1          Rabies - Day 13                                                     5          Rabies - Day 13                                                     9          Rabies - Day 12                                                     49         Rabies - Day 13                                                     86         Rabies - Day 11                                                     91         Rabies - Day 10                                                     105        Survived                                                            124        Rabies - Day 13                                                     134        Rabies - Day 11                                                     150        Rabies - Day 10                                      ______________________________________                                    

                  TABLE 4                                                          ______________________________________                                         Protection Afforded Cats by Vaccination with Inactivated                       Rabies Vaccine, Swine Testicle Cell Line Origin (Full Dose)                          Serum Neutralization Titer* Following                                                                   Status                                          Cat   Vac. (days)              Post-                                           No.   0      30     60   90   180  270  365  Challenge                         ______________________________________                                         NA6   Neg.   6166   64   102  64   100  64   Normal                            NF3   Neg.   407    646  407  151  263  64   Normal                            WK1   Neg.   8192   102  151  128  407  101  Normal                            WQ3   Neg.   2028   64   32   32   64   64   Normal                            SA2   Neg.   302    64   23   32   42   40   Normal                            WV4   Neg.   304    64   41   20   51   64   Normal                            NC1   Neg.   5248   256  128  324  214  64   Normal                            OG4   Neg.   1024   64   32   64   64   50   Normal                            NA1   Neg.   512    1024 1230 603  1622 256  Normal                            XB2   Neg.   1622   64   64   51   51   64   Normal                            SB3   Neg.   1024   64   91   128  214  64   Normal                            WD2   Neg.   1445   302  813  603  817  215  Normal                            NF4   Neg.   1660   76   64   128  54   54   Normal                            NA3   Neg.   8192   302  813  151  76   64   Normal                            NC4   Neg.   407    64   54   128  162  54   Normal                            BA1   Neg.   6457   302  128  151  407  215  Normal                            LD3   Neg.   256    56   32   46   32   16   Normal                            SB1   Neg.   8192   1622 1122 1738 1445 406  Normal                            SC3   Neg.   1318   102  162  151  427  101  Normal                            SA1   Neg.   512    64   16   64   107  64   Normal                            LC3   Neg.   407    64   81   107  128  40   Normal                            SG5   Neg.   4096   646  512  1230 1024 1024 Normal                            BC1   Neg.   214    102  151  427  646  304  Normal                            OF1   Neg.   407    54   38   64   256  64   Normal                            BB2   Neg.   6456   214  43   50   51   64   Normal                            Geo.                                                                           Mean  Neg.   1328   138  112  130  178  88                                     ______________________________________                                          *Titer expressed as reciprocal of serumneutralization end point.         

                  TABLE 5                                                          ______________________________________                                         Cat Controls - 20% Fox Brain Suspension of NYC Strain Street                   Rabies Virus (0.25 ml Bilateral Neck Muscles)                                  Challenge      Cat                                                             Dilution       No.        Observations                                         ______________________________________                                         1:30           22         Rabies - Day 15                                                     23         Rabies - Day 20                                                     24         Rabies - Day 14                                                     WQ2        Rabies - Day 13                                                     WL1        Survived                                                            25         Rabies - Day 20                                                     26         Rabies - Day 29                                                     28         Rabies - Day 35                                                     OF6        Rabies - Day 57                                                     SC2        Rabies - Day 29                                      1:60           ND1        Survived                                                            ND2        Rabies - Day 12                                                     NC3        Rabies - Day 31                                                     NA4        Survived                                                            NF2        Rabies - Day 17                                                     BF2        Rabies - Day 11                                                     BA3        Rabies - Day 34                                                     WS3        Rabies - Day 36                                                     OG2        Rabies - Day 13                                                     OF3        Rabies - Day 36                                      ______________________________________                                    

A further aspect of this invention is the preparation and use of combination polyvalent vaccines comprising vaccinal amounts of the adjuvanted inactivated rabies virus described herein and one or more canine or feline viruses. For example, feline vaccines comprising vaccinal amounts of modified feline rhinotracheitis virus, calicivirus, and/or panleukopenia virus combined with the inactivated rabies virus can be prepared. Such polyvalent vaccine will, preferably, contain from about 30% to about 70% total volume of the inactivated rabies virus, depending on the number of viruses in combination. An example of such a polyvalent feline vaccine contemplated by this invention comprises about 40% of the inactivated rabies virus, about 20% of feline rhinotracheitis virus, about 20% of calicivirus and about 20% of panleukopenia virus (all percentages based on total volume).

Likewise, canine vaccines comprising vaccinal amounts of distemper virus, canine adenovirus type 2 and para-influenza virus combined with the inactivated rabies virus can be prepared. Leptospira bacterin may also be added to such polyvalent vaccine. The polyvalent vaccine will, preferably, contain from about 30% to about 40% total volume of the inactivated rabies virus. An example of a polyvalent canine vaccine contemplated by this invention comprises about 40% of the inactivated rabies virus, about 40% of distemper virus and about 20% total of all other micro-organisms in the combination (all percentages based on total volume).

The polyvalent vaccines of this invention are administered parenterally, preferably by intramuscular injection. 

What is claimed is:
 1. An inactivated rabies virus vaccine capable of inducing immunity in feline animals without serious side effects comprising a vaccinal amount of inactivated HCP-SAD strain of rabies virus which has been adapted, before inactivation, to grow in swine testicle cell cultures, an adjuvant and a carrier therefor.
 2. The inactivated rabies vaccine of claim 1 wherein the rabies virus is passaged at least once in a swine testicle cell culture.
 3. The inactivated rabies vaccine of claim 2 wherein the rabies virus is passaged from 1 to about 25 times in a swine testicle cell culture.
 4. The inactivated rabies vaccine of claim 3 wherein the rabies virus is passaged from about 6 to about 12 times in a swine testicle cell culture.
 5. The inactivated rabies vaccine of claim 1, 2, 3 or 4 wherein the swine testicle cell culture is the NL-ST-1 swine testicle cell line.
 6. The inactivated rabies vaccine of claim 5 wherein the virus is inactivated with beta-propiolactone.
 7. The inactivated rabies vaccine of claim 5 wherein the adjuvant is aluminum hydroxide gel.
 8. A method of vaccinating feline animals comprising administering parenterally to said animals the inactivated rabies vaccine of claim 1, 2, 3 or
 4. 9. A method of vaccinating feline animals comprising administering parenterally to said animals the inactivated rabies vaccine of claim
 5. 10. The method of claim 8 wherein the vaccine is administered by intramuscular injection.
 11. The method of claim 9 wherein the vaccine is administered by intramuscular injection. 